Bottom Line:
Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo.A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth.Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Affiliation: Department of Radiation Oncology, Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACTEvidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Mentions:
We assessed whether the HUIV26 cryptic epitope could be exposed within the basal lamina of blood vessels in vivo. Unfixed biopsy sections from normal human skin were incubated with either activated or proMMP-2, HT1080 tumor–conditioned medium, or control buffer. The tissues were costained with Mab HUIV26 (green) and a polyclonal antibody directed to factor VIII–related antigen (red), a known marker of blood vessels. As shown in Fig. 2 A, blood vessels (red) from normal human skin were readily detected. Little if any of the cryptic HUIV26 epitope (green) was detected within the vascular basement membranes or the surrounding interstitial matrix from tissues treated with either inactive proMMP-2 or control buffer (Fig. 2 A, top). In contrast, tissues treated with either proteolytically active MMP-2 (Fig. 2 A, bottom left) or HT1080 tumor–conditioned medium (Fig. 2 A, bottom right) demonstrated exposure of the HUIV26 cryptic epitope, as indicated by colocalization (yellow) due to overlap of the exposed HUIV26 epitope (green) and factor VIII–related antigen (red). Together, these findings provide further evidence that the HUIV26 cryptic sites could be exposed by proteolytic activity in a physiological tissue.

Mentions:
We assessed whether the HUIV26 cryptic epitope could be exposed within the basal lamina of blood vessels in vivo. Unfixed biopsy sections from normal human skin were incubated with either activated or proMMP-2, HT1080 tumor–conditioned medium, or control buffer. The tissues were costained with Mab HUIV26 (green) and a polyclonal antibody directed to factor VIII–related antigen (red), a known marker of blood vessels. As shown in Fig. 2 A, blood vessels (red) from normal human skin were readily detected. Little if any of the cryptic HUIV26 epitope (green) was detected within the vascular basement membranes or the surrounding interstitial matrix from tissues treated with either inactive proMMP-2 or control buffer (Fig. 2 A, top). In contrast, tissues treated with either proteolytically active MMP-2 (Fig. 2 A, bottom left) or HT1080 tumor–conditioned medium (Fig. 2 A, bottom right) demonstrated exposure of the HUIV26 cryptic epitope, as indicated by colocalization (yellow) due to overlap of the exposed HUIV26 epitope (green) and factor VIII–related antigen (red). Together, these findings provide further evidence that the HUIV26 cryptic sites could be exposed by proteolytic activity in a physiological tissue.

Bottom Line:
Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo.A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth.Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Affiliation:
Department of Radiation Oncology, Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACTEvidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.